Glioblastoma is the most common subtype of primary brain tumors in adults. These tumors are highly invasive. Median survival after diagnosis is approximately 12 months. Chemotherapy with nitrosourea, methylating agents such as procarbazine or temozolomide, or other agents is effective and can prolong survival. However, a significant obstacle to the treatment has been the hematopoietic toxicity of chemo- therapeutic agents like 1,3-bis(2-chloroethyl)-1 -nitrosourea (BCNU) and temozolomide, which has limited the ability to dose-escalate the drugs. This hematopoietic toxicity is even more pronounced when BCNU or temozolomide are combined with O6-benzylguanine (O6-BG), an agent which inhibits O6-alkylguanine DNA alkyltransferase, a major resistance mechanism in gliomas. We have recently demonstrated in a clinically relevant large animal model that genetic modification of hematopoietic stem cells with the P140K mutant of methylguanine methyltransferase (MGMT) results in chemoprotection and the ability to administer higher doses of temozolomide and BCNU when given in combination with O6-BG. Based on these findings we hypothesize that chemoprotection with MGMT (P140K) will allow the administration of higher doses of temozolomide and thus potentially improve the efficacy of this drug in the treatment of patients with high-grade gliomas. Since efficient engraftment of gene-modified cells is important for the success of this strategy, we propose a 2-part approach. In the first part, we propose a dose-escalation of temozolomide with a fixed dose of BCNU to enhance engraftment of gene-modified chemoprotected peripheral blood stem cells, followed by fixed doses of temozolomide with O6-BG. The initial dose of BCNU and temozolomide will be based on the maximum tolerated dose (MTD) published by the North American Brain Tumor Consortium. This approach will allow us to maximize engraftment of chemoprotected cells and thus also maximize the ability to dose-escalate temozolomide. The initial dose of temozolomide will be based on the published MTD for this combination. In the second part of the study, we will use the MTD of BCNU and temozolomide with support of chemoprotected peripheral blood stem cells and perform a dose-escalation of temozolomide with O6-BG. Thus, this study will address the safety, feasibility, and tumor response of dose-intensive chemotherapy in combination with chemoprotected autologous stem cells in patients with glioblastomas. In addition, we will perform a comprehensive molecular analysis of the gene-modified cells which will provide highly relevant information for stem cell gene therapy studies. Research proposed in this study could, therefore, significantly improve the treatment and survival of patients with glioblastoma and also provide important safety and feasibility information for stem cell gene therapy applications for other diseases such as genetic disorders and AIDS.